1,720,962 research outputs found
Role of Fusarium graminearum cerato-platanin and hydrophobin proteins in fungal growth and plant infection
Full text linkCerato-platanins (CPPs) and hydrophobins (HPs) are small secreted non-catalytic cysteine-rich proteins typical of filamentous fungi. CPPs are possibly localized in the fungal cell walls, they are similar to plant expansins having carbohydrate binding/loosening properties with a non-enzymatic mechanism and can have phytotoxic activity. HPs, after being secreted as monomers from the hyphal apexes, cover the fungal surfaces with a hydrophobic layer and may be involved in several processes such as formation of fungal aerial structures, attachment to hydrophobic surfaces, interaction with the environment and protection against the host defense system by masking the fungal cell wall. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease (FHB) of wheat and other cereal grains, contains two genes putatively encoding for proteins with a CP domain, named fgcpp1 and fgcpp2, and five genes encoding for HPs, named FgHyd1-5. In order to verify their contribution during plant infection and fungal growth, single and multiple gene knock-out mutants were produced and characterized. Besides, the two F. graminearum CPPs (FgCPPs) were heterologously expressed to investigate their activity.
The FgCPPs seem to be dispensable for fungal virulence but protect fungal cell wall polysaccharides from enzymatic degradation. The FgCPPs show a strong ability to reduce, mainly by a non-enzymatic mechanism, the viscosity of carboxymethyl cellulose (CMC), with higher affinity for substrates with medium/high viscosity and favour fungal cellulase activity. The observation that the double knock-out mutant ∆∆fgcpp1,2 grown on CMC produced more cellulase activity than the wild type suggests that the higher enzymatic activity produced by the mutant could compensate during infection for the absence of the FgCPPs activity on plant cellulose.
The F. graminearum Hyd2 and Hyd3 are responsible of the hydrophobicity of aerial hyphae and are involved in adhesion of conidia to the host surface during the early stages of the infection process, as shown by the reduced virulence of the ∆hyd2 and ∆hyd3 on Triticum aestivum observed by spray inoculation. Interestingly, triple ∆hyd1,2,3, ∆hyd2,3,4 and ∆hyd2,3,5 mutants produced a reduced number of mature perithecia and showed defects at the cell wall level, being significantly more inhibited than the wild type by β-1,3-glucanase and more susceptible to tebuconazole, an ergosterol biosynthesis inhibitor fungicide.
Based on our results, FgCPPs and FgHyds could be used as targets of new molecules in innovative disease management strategies aimed at increasing fungal susceptibility to plant defense proteins or at reducing fungal ability to adhere to host plant tissues. Furthermore, the ability of FgCPPs to loosen CMC and favor cellulase activity could make these proteins suitable for future potential applications in biofuel production using cellulose rich tissues as substrate
The Fusarium graminearum cerato-platanins loosen cellulose substrates enhancing fungal cellulase activity as expansin-like proteins
No full textCerato-platanin proteins (CPPs) are small non-catalytic, cysteine-rich hydrophobic proteins produced by filamentous fungi. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease of wheat and other cereal grains, contains two genes putatively encoding for CPPs. To better characterize their features, the two FgCPPs were heterologously expressed in Pichia pastoris. The recombinant FgCPPs reduced the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC). The same effect was not observed on other polysaccharide substrates such as chitin, 1,3-β-glucan, xylan and pectin. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs are trapped by cellulose and not by chitin, thus suggesting that these proteins interact with cellulose. A double knock-out mutant deleted of both FgCPPs encoding genes produce smuch more cellulase activity than the corresponding wild type strain when grown on CMC, likely compensating the absence of FgCPPs. This result prompted us to investigate a possible synergistic effect of these proteins with fungal cellulases. The incubation of FgCPPs in the presence of a fungal cellulase (EC 3.2.1.4) determines an increased enzymatic activity on CMC, filter paper and wheat cell walls. The observation that FgCPPs act with a non-hydrolytic mechanism indicates that these proteins favor fungal cellulase activity in an expansin-likemanner. Though the disruption of the FgCPP genes had no demonstrable impact on fungal virulence, our experimental data suggest their probable involvement in virulence, thus we refer to them as accessory virulence genes. Our results suggest also that the FgCPPs could be exploited for future biotechnological application in second-generation biofuels production on lignocellulosic biomasses rich in cellulose. Finally, we demonstrate that FgCPPs act as elicitors of defense responses on Arabidopsis leaves, increasing resistance to Botrytis cinerea infections
Twenty years of research on cerato-platanin family proteins: clues, conclusions, and unsolved issues
No full textTwenty years of research on cerato-platanin family proteins (CPPs) have led to some clear conclusions: CPPs are exclusively present in the fungal kingdom and possess an outstanding capacity to stimulate the immune system of plants. Recent discoveries have highlighted remarkable structural and functional similarities between CPPs and expansins, a class of non-enzymatic proteins found in both plants and microbes possessing loosening ability on the cell wall structure. Nevertheless, the determination of a biological role for CPPs in fungi is becoming a complicated puzzle to solve, since experimental data are often divergent and point to functional diversification. A general consensus appears however possible: CPPs from pathogenic and beneficial fungi may be considered as microbe-associated molecular patterns (MAMPs) and likely play a dual role, exerting functions in the fungal cell wall and/or in plant colonization. In this review, which celebrates 20 y of research on CPPs, we trace the history of these proteins and highlight experimental evidence and still unsolved issues
Fusarium graminearum cerato-platanin proteins weaken cellulosic materials and enhance cellulase activity in an expansin-like manner
No full textCerato-platanin proteins (CPPs) belong to a family of small secreted non-catalytic fungal proteins with phytotoxic activity. CPPs have been recently classified as expansin-like proteins because of structural and functional features related to plant expansins, small secreted proteins able to loosen and disrupt the non-covalent bonding networks of plant cell wall polysaccharides without enzymatic activity. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease of wheat and other cereal grains, contains two genes putatively encoding for CPPs (FgCPPs). To characterize their role, the two proteins have been heterologously expressed in yeast. Enzymatic assays have shown the ability of the recombinant FgCPPs to reduce the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC) mainly with a non-enzymatic activity. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs seem trapped by cellulose and not by chitin, thus suggesting that they could interact with cellulose. The incubation of CMC with a cellulase in presence or absence of the two recombinant proteins has shown that the FgCPPs enhance cellulase activity. A double knock-out mutant deleted of both FgCPPs encoding genes produces higher cellulase activity when grown on CMC, thus suggesting that the absence of FgCPPs forces the fungus to produce more cellulase activity to compensate for the lack of expansin-like activity. Finally, the preliminary demonstration that the FgCPPs act also loosening filter paper, a natural insoluble cellulose, could suggest a possible future biotechnological application in second-generation biofuels production from agricultural lignocellulosic biomasses rich in cellulose
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Characterization of some industrially relevant polysaccharide monooxygenases and related enzymes
No full textINTRODUCTION AND AIMS
By 2030, global rice production is expected to increase to meet the demand of a growing world population. However, rice is severely affected by blast disease, caused by the fungus Magnaporthe oryzae, which can reduce total annual rice production by 10-30% and, if not controlled, can cause complete loss of production.
In the early stages of the infection process, M. oryzae forms an infection structure called appressorium to break the plant cuticle and it expresses many polysaccharide and lignin degrading enzymes: among them, polysaccharide monooxygenases (PMOs) degrade polysaccharides by an oxidative mechanism and could be important virulence factors for the fungus.
The first objective of the project is to identify M. oryzae PMOs and related enzymes active on polysaccharides and lignin essential for pathogenesis on rice. A screening of natural molecules or inhibitor proteins will then be performed to identify those effective in inhibiting the essential fungal enzymes, thus reducing growth and infection rate of M. oryzae. The final aim of this objective is to develop new methods to control rice blast disease in order to increase food production.
Rice straw is the main by-product of rice-producing areas and is a potential resource for biofuel production. Worldwide, about 400 million tons of rice straw are annually produced, with about 30-40 million tons/year in Vietnam alone, but more than 95% is burned, resulting in hazardous airborne emissions. Several polysaccharide and lignin-degrading enzymes like PMOs have been applied in the biofuel industry. When mixed to the typical cellulose-hydrolyzing enzymes, PMOs could dramatically change the enzyme technology involved in biomass degradation, reducing biofuel production costs. However, the activity of PMOs on rice straw needs to be further characterized. The second objective of the project is to heterologously express the M. oryzae PMOs and related enzymes to characterize their enzymatic activities on polysaccharides and lignin, with the aim of developing new enzyme mixtures for rice straw degradation to be used in biofuel production.
Aim 1: Characterization of the pathogenetic mechanism of Magnaporthe oryzae to develop new rice blast disease control measures
Candidate M. oryzae genes encoding PMOs and related polysaccharides and lignin hydrolyzing enzymes have been identified by an in silico analysis of the fungal genome, completely sequenced. Their expression during the infection process, and particularly during appressorium formation, have been characterized by transcriptomic analysis.
Reverse genetics approach: the knock-outs of the most expressed genes (two PMOs, two beta-1,3-glucanases, one ligninase, one chitin synthase and one chitinase) are in progress by targeted homologous recombination.
Screening of mutants: infection of rice seedlings and plants will be performed to determine the contribution of the fungal enzymes of interest to fungal virulence.
The ability of mutants to form appressorium will also be observed by light microscopy.
Natural bioactive compounds and protein inhibitors potentially inhibiting the M. oryzae enzymes shown to play important roles for fungal virulence will be identified by in silico simulation of protein structures. A screening by in vitro bioassays for their ability to inhibit or reduce M. oryzae growth and its virulence on rice seedlings and plants will then be performed. Bioassays will also be performed to evaluate the inhibition potential of bioactive compounds and protein inhibitors on appressorium formation. Effective inhibitors of the target enzymes will then be tested on rice field trials. Rice transgenic plants expressing proteins able to inhibit some target enzymes of M. oryzae will be also created.
Aim 2: Application of Magnaporthe oryzae PMOs and related enzymes to rice straw degradation and biofuel production
Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species. They have recently been found to degrade recalcitrant polysaccharides, including chitin, cellulose and starch with an unprecedented oxidative mechanism. Polysaccharides are known to be degraded by hydrolytic enzymes, termed collectively as glycoside hydrolases (GHs). The available structures of PMOs reveal a conserved fold and a highly conserved monocopper active site on a flat protein surface. PMOs likely act directly on the substrate surface, bypassing the energy-intensive step of removing polysaccharide chains from the insoluble substrates required in GHs. The new ends created by PMOs can be subsequently processed by various GHs, including exo- and endo-glucanases, resulting in enhanced overall polysaccharide degradation.
Genes encoding for M. oryzae PMOs and related polysaccharides and lignin degrading enzymes, previously identified by transcriptomic analysis, will be obtained from M. oryzae RNA from infected rice tissues and the corresponding enzymes of interest will be heterologously expressed using the Pichia pastoris yeast system.
The recombinant PMOs will then be purified and characterized at their mononuclear copper active site by UV/Vis, rRaman, Electron Paramagnetic Resonance (EPR) and X-Ray Absorption (XAS).
The recombinant enzymes will also be purified at a larger scale (pilot scale) and tested for their enzymatic activity by bioassays performed on different substrates. Since PMOs, like other glycoside hydrolases, have different activities on different types of materials, the purified recombinant enzymes will be tested alone or in mixture in order to optimize the digestion of rice straw. In addition, optimization of ratios of M. oryzae PMOs and commercialized polysaccharide hydrolysis enzymes for degradation of rice straw will be performed.
The final aim is the development of a novel rice straw degrading enzyme technology.
PERSPECTIVES
The expected results, that is the characterization of the role and enzymatic activities of M. oryzae PMOs and related enzymes in order to identify new methods to control rice blast disease and to develop new enzyme mixtures for rice straw degradation and biofuel production, will be exploited for production of scientific publications, dissemination purposes and transfer of new agricultural and industrial technologies. In particular, the PMOs or polysaccharide hydrolase enzymes identified in the project will be tested at the trial scale at bioethanol production factories; the bioactive compounds identified in the project and able to inhibit or control rice blast disease will be applied in rice field trials. The expected impact and benefits of these activities are enhancing the use of rice straw for bioethanol production, with consequent reduction of dependence on fossil fuels and rice straw burning, and the effective inhibition or control of the rice blast disease, with consequent increase of rice productivity and improvement of rice farmers’ economic conditions. Another expected result of the proposed research is training several students: in particular, visits at the Vietnam and Italian laboratories will enable the training of young researchers and the future development of new research objectives aimed at improving the agricultural and industrial systems of both countries
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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